The effect on energy metabolism of a 6-h prolongation of the conventional 12-h overnight fast was examined in 9 healthy subjects and in 7 patients with non-insulin-dependent diabetes mellitus. Plasma glucose concentration decreased by 7 and 23%, in control and diabetic subjects, respectively. In control subjects, the fall in plasma glucose was associated with a slight but significant fall in plasma insulin and a rise in plasma free fatty acid concentrations. During this same period, the rates of plasma free fatty acid oxidation, measured by infusion of [14C]palmitate, and net lipid oxidation, measured by indirect calorimetry, increased in normal subjects by 55 and 76%, respectively; the rate of glucose oxidation measured by indirect calorimetry decreased by 37%. In the diabetic patients, the free fatty acid oxidation rate was enhanced already after 12 h of fasting compared with controls (2.06 vs 1.30 mumol.kg-1.min-1; p less than 0.05) and did not change significantly during the 6-h observation period. After 18 h of fasting, the rate of plasma free fatty acid oxidation was similar in control and diabetic subjects. The data thus emphasize the need for strict standardization of the overnight fasting period for metabolic studies, and demonstrate the difficulties in comparing basal rates of substrate oxidation between healthy and diabetic subjects.